Significance: The sarcoplasmic reticulum Ca ²⁺ -ATPase (SERCA)/phospholamban complex regulates cardiac muscle contractility by controlling Ca ²⁺ transport from the cytosol to the lumen of the sarcoplasmic reticulum. By mapping the interactions between these two membrane proteins, we found that SERCA function depends on the equilibria between transient conformational states of phospholamban. Phosphorylation of phospholamban shifts the equilibria, enhancing SERCA function. This mechanism explains why tuning phospholamban’s structural dynamics can modulate SERCA function and may aid in designing innovative therapeutic approaches to heart failure.

• Structure of phosphorylated phospholamban solved by hybrid NMR method • Phosphorylated phospholamban exhibits motions in the cytoplasmic domain • Phosphorylated phospholamban does not conduct ions selectively • Oligomerizaion is a mechanism to control Ca²⁺-ATPase inhibition